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. 2015 Aug 17;156(11):3961–3970. doi: 10.1210/en.2015-1321

Figure 3.

Figure 3.

The role of ASBT in bile acid–induced GLP-1 release and intracellular Ca2+ changes. Expression of Asbt was determined in homogenized intestine segments (A) and FACS-isolated L-cell populations (B). Expression of Asbt (Slc10a2) in control cells (□) and L-cells (■) was determined relative to that of β-actin by qRT-PCR. Up SI, upper 10 cm of the small intestine; low SI, lower 12 cm of the small intestine; LI, large intestine. Mean ΔCT and upper SEM were calculated from 3 independent experiments and are presented as 2ΔCT. Statistical tests were assessed on nontransformed data using a one-way ANOVA and post hoc Bonferroni test (A) or two-tailed t tests (B) (*, P < .05; **, P < .01). C–F, Mixed lower small intestinal cultures of mice expressing GCaMP3 in L-cells were perfused with standard saline containing 10 mmol/L glucose and additions as indicated. C, Representative trace depicting GCaMP3 fluorescence (Fl) in response to TDCA (100 μmol/L) applied in standard and low (5.6 mmol/L) Na+ saline and 30 mmol/L KCl. D, Mean increase in GCaMP3 fluorescence over baseline (Fl/Fl0) for cells recorded as in C. Cells that did not respond to any stimuli (all responses <1.1-fold) were excluded (2 of 14). Results are means ± SEM (n = 12). Statistical significance from basal was determined on log10-transformed data via a one-sample t test (**, P < .01). E, Representative trace depicting a calcium response to TDCA (10 μmol/L) with and without the ASBT inhibitor (ASBT-I; 10 μmol/L). F, Mean ± SEM data are recorded as in E (n = 4). Statistical analysis was performed as in D (***, P < .001 from basal). G, Lower small intestinal cultures were incubated for 2 hours in saline solution containing 10 mmol/L glucose and stimuli as indicated. Test agents were TDCA (100 mymol/L), ASBT-I (10 μmol/L), and 10 μmol/L forskolin plus 10 μmol/L IBMX (F/I). GLP-1 release is expressed as a percentage of content. Results are means ± SEM of n = 12 wells, with 4 wells originating from a single mouse performed in parallel. Statistical differences were determined using a one-way ANOVA and post hoc Bonferroni test on log10-transformed data (**, P < .01; ***, P < .001, compared with basal).